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327
1 OF BETACYANIN PIGMENTS
STABILITY
FROM RED PURPLE PITAYA FRUIT (Hylocereus polyrhizus) :
INFLUENCE OF PH, TEMPERATURE, METAL IONS AND ASCORBIC ACID
Tang, C.S and Norziah M.H*
School of Industrial Technology, Food Technology Division,
Universiti Sains Malaysia, 11800 Penang, Malaysia.
Received 30 May 2007; Accepted 30 July 2007
ABSTRACT
Betacyanin pigments from red-purple pitaya fruit (Hylocereus polyrhizus) could be an attractive source of red
colourant for food application. This paper presents results on the extraction of betacyanin pigments from pitaya fruits
grown locally in Malaysia. Both the flesh of the fruit and its mesocarp were investigated and it was found that the
flesh had higher pigment contents compared to its peel component. The concentration of betacyanins expressed as
betanin equivalents per 100 g of flesh and peel were 10.1 0.6 mg and 6.7 0.2 mg, respectively when 80%
methanol was used. The stability of betacyanin pigments were investigated at different pH, temperature and in
2+
2+
presence of different concentrations of metal ions (Cu and Fe ) and ascorbic acid. The results showed that the
pigment was most stable at pH range between 5 and 6. However, it forfeited its stability to the heat induced at
2+
2+
elevated temperatures. Metal ions (Cu and Fe ) proved to be capable of accelerating betacyanin degradation, with
2+
Cu exhibiting the greatest effect. By contrast, supplementation with ascorbic acid could enhance the pigment
stability against the detrimental effects caused by pH, temperature and metal ions. Nevertheless, if the concentration
of ascorbic acid exceeds 0.7 %, it may change its role from pigment stabilizer to become a pro-oxidant.
Keywords: Betacyanin, pigments, pitaya fruit, Hylocereus polyrhizus, ascorbic acid
INTRODUCTION
Natural colourants from plant sources are receiving
growing interest from both food manufacturers and
consumers in the continuing replacement of synthetic
dyes [1,2]. However, replacing synthetic dyes with
natural colorants offers a challenge because the colour
and stability of plant pigments are dependent on several
factors, including structure and concentration of the
pigment, pH, temperature, light intensity, presence of,
metallic ions, enzymes, oxygen, ascorbic acid, sugars
and their degradation products, among others [3].
Identifying stable aqueous colorant extracts (e.g. fruit
and vegetable juices) is attractive because their GRAS
(Generally Recognized As Safe) status makes them
easily commercialized.
The fruits of Hylocereus species, known as red
pitaya or pitahaya, which means the scaly fruit, in Latin
America, belong to the cacti family which is native to the
tropical forest regions of Mexico and Central and South
America. In cacti, the most important fruit pigments are
the red-violet betacyanins and the yellow betaxanthins
which belong to the betalain pigments [4]. Producing a
deep purple-coloured flesh comparable to red beet [5] or
amaranth [6], betacyanins from purple pitaya (H.
polyrhizus) has recently attracted interest as a potential
alternative for red- beet.
In the present study,
betacyanin from purple pitaya (H. polyrhizus) was
extracted using several solvents. However, the lower
stability of natural plant pigments against environmental
* Corresponding author. Tel: 04-6532222/Fax : 04-6573678
Email address : norziah@usm.my
328
1
give the highest extraction yield. The homogenized
fruit
flesh or peel was thawed prior to extraction. For pigment
extraction from fruit flesh, solvents used were 80 %
acetone, 80 % methanol and water. However, for peel
component, only 80 % acetone and 80 % methanol were
used for pigment extraction. During pigment extraction, 1
part of the sample (fruit flesh or peel) was shaken with 2
parts of solvent for a few minutes. It was allowed to
stand for 15 min. Then, the seeds, raw fibres, mesocarp
fibres and mucilagenous material were separated from
the pigment extract by centrifugation at 3500 rpm for 15
min and filtration. The residue was re-extracted with the
selected solvents in the same manner. The filtrate was
0
combined with that obtained earlier and stored at -20 C
prior to betacyanin pigments quantification.
Quantitation of betacyanins
Red pitaya pigment content was measured in a
similar way to that described by Cai et al [6], with slight
modification. Quantification of betacyanins was carried
out applying the equation Bc = [(A x DF x MW x 1000 /
x l)] where Bc is the betanin equivalents in mg/L, A is the
absorption value at the absorption maximum (max = 536
nm), DF is the dilution factor, MW is the molecular
weight of betanin (550 g/mol), is the molar extinction
coefficient of betanin (60,000 l/mol x cm), and l the
pathlength of the cuvette. All determinations were
performed in triplicate.
Pigment stability studies
Based on the yield of betacyanin content, water was
selected as solvent for pigment extraction for the stability
studies. The stability of betacyanin aqueous-based
pitaya fruit extracts was evaluated under different pH
o
values (2 to 10), temperature (25 to 75 C), in presence
2+
2+
of metal ions such as Cu and Fe (concentrations
ranging from 0 to 150 ppm) and ascorbic acid
(concentration 0 to 1.6 ppm). The pigment retention
used to indicate colorant degradation as affected by
these factors was calculated in each case after 12 hrs
under the experimental conditions described above.
Pigment retention (%) was calculated by the formula
[betacyanin content after storage time interval /
betacyanin content at zero storage time] x 100. The
effects of pH, metal ions and ascorbic acid on pigment
o
retention were studied at 25 C with sample inside
capped vials covered with aluminium foil and sealed with
parafilm. For the effects of temperature studies, test
samples at pH 5.0 were immersed in a water bath and
kept at various temperatures (25, 30, 35, 40, 45, 50, 55,
o
60, 65, 70, 75 C) for 12 h. The test samples (10 mL)
were prevented from the exposure of light and
atmosphere as both factors could cause additional
negative
effects
to
the
pigments.
Triplicate
measurements will be carried out.
329
100
120
100
Pigment retention (%)
80
60
40
20
80
60
40
20
0
0
0
10
12
0.2
0.4
0.6
pH
1.2
1.4
1.6
90
90
80
80
70
70
0.8
60
50
40
30
60
50
Cu2+
Fe2+
40
30
20
20
10
10
0
0
0
10
20
30
40
50
60
70
80
Temperature (deg C)
20
40
60
80
100
120
140
160
330
120
120
100
100
80
60
40
5 deg C (control)
25 deg C (control)
20
80
60
40
20
5 deg C (control)
25 deg C (control)
0
0
10
12
14
16
18
20
22
24
26
28
No of days
Lightness
a*
b*
Chroma*
Hue angle
52.26
82.13
-27.36
86.57
341.57
Lightness
a*
b*
Chroma*
Hue angle
52.26
82.13
-27.36
86.57
341.57
Lightness
A*
B*
Chroma*
Hue angle
52.26
82.13
-27.36
86.57
341.57
Lightness
a*
b*
Chroma*
Hue angle
52.26
82.13
-27.36
86.57
341.57
10
12
14
16
18
22
24
26
28
thus
measurements
could
not
be
made.
Supplementation with ascorbic acid increased storage
o
o
stability at 5 C (91.6%) than at 25 C (70.9%). Samples
o
at 5 C with addition of ascorbic acid did not show a
decrease in pigment content for about 2 weeks
compared to samples with ascorbic acid which showed a
small reduction in pigment retention after about 5 days.
This indicated that there is a stabilising effect of ascorbic
acid on pigment retention. Fig 6 and Table 2 give results
on the colour properties of pigment preparations.
Greater colour retention at both temperatures in samples
with ascorbic acid compared to samples without ascorbic
20
No of days
21
28
57.12
72.64
22.27
75.98
342.96
58.05
69.64
-21.27
72.82
343.01
52.31
81.61
-32.03
87.67
338.57
52.69
80.09
-32.70
86.51
337.79
53.12
72.85
-35.27
80.94
334.16
55.54
69.81
-34.28
77.77
333.84
CONCLUSION
331